Q5 mutagenesis

GST-, MBP-, and his-tagged Par-3 constructs, GST-aPKC PBM, and his aPKC kinase domain-PBM (residues 259–606) were cloned as previously described (10 (link)) using Gibson cloning (New England Biolabs), Q5 mutagenesis (New England Biolabs), or traditional methods. In addition to an N-terminal MBP tag, the Par-3 PDZ1-APM (residues 309–987) construct also contained a C-terminal his-tag. Par complex components (aPKC and his-Par-6) were cloned into pCMV (human cytomegalovirus) as previously described (10 (link), 23 (link)). Please see the Key resources table for additional information on specific constructs.

Full-length human RIG-I was previously cloned into a pET SUMO expression vector (19 (link)). This construct contains an N-terminal 6-His tag, followed by a SUMO tag and the complete human RIG-I protein sequence. A serine residue was also added between the SUMO tag and RIG-I to ensure efficient tag cleavage.
In this study, we added the amino acid sequence MPCCPGCCGS following RIG-I residue 190. This sequence was added using Q5 mutagenesis (New England Biolabs) according to the manufacturer’s instructions. AzF was incorporated by amber stop codon suppression. Thus, the wild-type amino acid Glu⁴⁹⁴ was mutated to an amber stop codon (UAG) by the QuikChange Site-Directed Mutagenesis Kit (Agilent) according to the manufacturer’s recommendations.

Plasmids used in this study are listed in Table S1. pIG1783-Smkin3-gfp was created by restricting the plasmid pIG1783 with NcoI. Smkin3 was amplified via PCR with overhangs at the 5′ and 3′ ends containing recognition sites for NcoI. Ligation was performed with T4 DNA ligase. For phospho-mimetic and phospho-deficient strains, plasmid pIG1783-Smkin3-gfp was used for Q5 mutagenesis (New England Biolabs). With specific primers (Table S3), we generated eight plasmids containing phospho-mimetic and phospho-deficient mutations (Table S1).

pAR008 allows assembly of a custom CRR coding sequence into a truncTALE (Tal2h) backbone coding sequence using our previously described Golden Gate kit for custom TAL effector construct assembly (Cermak et al., 2011 (link)). The vector was created in two steps with the NEBuilder HiFi DNA assembly kit (New England Biolabs, Ipswich, MA). First, Golden Gate entry vector pTAL1 (Cermak et al., 2011 (link)) was used as a template in a PCR reaction with primer pairs B1778/B1779 and B1781/B1782. The resulting intermediate vector was used as template in a second round of PCR with primer pair B2149/B2150, and Gibson assembled with coding sequence for the Tal2h N-terminal region amplified from genomic subclone pYH2 using primer pair B2147/B2148. Entry vector pAR009 is a version of pTAL1 that contains a silent mutation to abolish the SphI site in the tal1c 3′ end, modified using Q5 mutagenesis (New England Biolabs) with primer pair B2171/B2172. Entry vector pAR012 is pAR008 further modified with a stop codon just prior to the coding sequence for the candidate NLS of Tal2h (RKRKSHD). It was generated in a Q5 mutagenesis reaction with primer pair B2168/B2169. An SphI site and an AatII site in each of these vectors can be used together to shuttle CRR coding sequences from existing TAL effector clones.

A complete list of plasmids used or generated in this study is provided in S2 Table. CRISPR/Cas9 plasmids were adapted from the pSAG1:CAS9,U6:sgUPRT plasmid previously generated by our lab [33 (link)]. The guide RNA of the plasmid was modified to target the AAH2 5’ UTR by Q5 mutagenesis (New England Biolabs, Ipswich, MA), creating the plasmid pSAG1:CAS9,U6:sgAAH2. A second guide RNA expression cassette targeting the AAH2 3’ UTR was inserted into the same plasmid backbone by traditional cloning steps to create the CRISPR/Cas9 AAH2 double cut plasmid pSAG1:CAS9,U6:dgAAH2. The same plasmid backbone was similarly adapted to target the AAH1 5’ and 3’ UTRs (pSAG1:CAS9,U6:sgAAH1 and pSAG1:CAS9,U6:dgAAH1). The pSAG1:CAS9,U6:sgUPRT plasmid described previously [33 (link)] was also modified to create a double-cutting CRISPR/Cas9 plasmid targeting the HXGPRT gene pSAG1:CAS9,U6:dgHXGPRT [34 (link)]. Plasmids used to generate the Δaah2 knockout using the HXGPRT selectable marker to replace the gene in the ME49Δhxg::Luc strain [31 (link)], and to restore expression of AAH2 were described previously [24 (link)]. Plasmids used to generate the Δaah1 mutant by replacement with the selectable marker DHFR-Ts, and to complement expression with a cDNA construct targeted to the uracil phosphoribosyl transferase (UPRT) locus, were created using Gibson assembly (New England Biolabs).

To build tet-OFF mutants of TgCrks and TgCyclins, the 5’ end of the corresponding gene was amplified (primers used are listed in S1 Table), digested with BglII/XhoI or BamHI/XhoI and ligated into the promoter replacement vectors, ptetO7sag4-myc-DHFR-TS or ptetO7sag4-HA_3X-DHFR-TS [24 (link), 71 (link), 72 (link)]. The vector ptetO7sag4-HA_3X_DHFR-TS was created by epitope swap on the ptetO7sag4-myc_DHFR-TS plasmid (Q5 mutagenesis, New England Biolabs). The resulting TgCrk and TgCyclin tet-OFF constructs were linearized, introduced into the Tati-RHΔku80 [40 ] strain and selected for pyromethamine resistance. Successful recombination into the locus was verified by PCR and individual transgenic clones were screened by IFA. To test conditional regulation, the tet-OFF mutant clones were grown with or without 1μg/ml ATc and protein expression level of the tet-OFF factors was analyzed by Western Blot analysis and IFA. Double-tagged transgenic lines were established by sequential electroporation and corresponding drug selection.